Bartonella bacilliformis (Bb) is a highly virulent, sand fly-borne, Gram-negative bacterium that causes Carrin?s disease in humans. Carrin?s disease is emerging in endemic regions of the Andes and expanding into historically non-endemic areas of Ecuador, Colombia and Peru. The at-risk population is currently ~1.7 million people in a 56,000 square-mile area. Incidence rates of 12.7/100 person years have been reported in endemic regions. Bb infections can be life-threatening, with fatality rates of 40-88%, if left untreated. Pediatric populations are especially at high-risk. In non-endemic regions, the disease often manifests as an acute illness with an ~80% reduction in erythrocyte hematocrit (Oroya fever), whereas in endemic regions, angiomatous skin lesions (verruga peruana) and chronic bacteremia prevail, effectively creating a human reservoir. Little is known about Bb?s molecular pathogenesis and relationship with its phlebotomine sand fly vector, Lutzomyia verrucarum. We hypothesize that small, noncoding RNAs (sRNAs) play key roles in optimizing Bb?s ability to survive and replicate in human cells and sand flies via transcriptional and post-transcriptional regulation. Bb?s natural history offers an exceptional model to examine regulatory roles of sRNAs, as the pathogen is frequently subjected to dramatic shifts in temperature, pH, and disparate chemistries of the extracellular and intracellular niches of the sand fly vector and human host, respectively. Our long-term goal is to elucidate how Bb regulates the adaptive responses necessary for infection, with overarching objectives of: a) generating information that can be applied towards the eventual eradication of Carrin's disease and b) providing a model of sRNA-mediated regulation of survival genes (e.g., stress response, metabolism, virulence, etc.) in Bartonella and other arthropod-borne, intracellular pathogenic bacteria. The hypothesis will be addressed by three specific aims. In aim 1, we will characterize Bb?s baseline transcriptome (sRNAs and mRNAs) during axenic growth using RNA-Seq. Differential transcription as a function of relevant environmental cues will also be explored. Second, we will compare transcriptomes of Bb grown axenically to those infecting human vascular endothelial cells and erythrocytes. These results will allow us to identify infection-specific transcripts produced in the intracellular niche. In aim 2, we will characterize the transcriptome expressed during infection of sand flies. From these results, we can identify infection-specific RNAs produced during extracellular growth in the insect vector. In aim 3, we will analyze functions of the two dominant, infection-specific sRNAs identified above; one produced in host cells and a second in sand flies. First, we will generate respective sRNA mutant and overexpression strains by genetic manipulation. Second, strains will be analyzed for growth and survival during infection, and their transcriptomes analyzed to identify potential targets. The mRNA targets of the dominant sRNAs will be verified by EMSA and the results used to formulate the respective sRNA-mediated regulatory networks. These results will define the functions for two dominant, ?infection-specific? sRNAs of Bb.